Thermo Fisher Scientific Rat IL-4 Matched Pair User guide

Type
User guide
Rat IL-4 Matched Pair
Module Set for the development of an ELISA for quantitative detection of rat IL-4
Catalog Number BMS628MST
Pub. No. MAN0016901 Rev. A.0 (30)
WARNING! Read the Safety Data Sheets (SDSs) and follow the
handling instructions. Wear appropriate protective eyewear,
clothing, and gloves. Safety Data Sheets (SDSs) are available
from thermosher.com/support.
Read before opening
Some vials contain small quantities of material, therefore
centrifuge before use.
This set of reagents is intended for use by persons experienced in
the use of immunoassays. It is not suitable for use by
inexperienced personnel.
A sample protocol is included, but the protocol provided is a
guideline. The type of substrate as well as all other reagents not
included in the Module Set may inuence assay performance.
Reagents provided
1 vial (1.1 mL) monoclonal Coating Antibody to rat IL-4 (100 µg/mL)
1 vial (55 µL) Biotin-Conjugate anti-rat IL-4 monoclonal antibody
1 vial (11 µL) Streptavidin-HRP
1 vial rat IL-4 Standard protein lyophilized, 2 ng/mL upon
reconstitution
2 vials (50 mL) Sample Diluent
Storage instructions
Store the kit components at 2°C to 8°C. Immediately after use
remaining reagents should be returned to cold storage (2°C to 8°C).
Aliquot reagents for repeated use at later dates. Reagents are labeled
with expiration date. For specic storage instructions see also
“Preparation of immunological reagents“ on page 2.
Samples should be aliquoted and must be stored frozen at -20°C to
avoid loss of bioactive rat IL-4.
Reagents and materials not provided
Microwell plate
Buers and solutions (see “Preparation of buers and
solutions“ on page 1 for preparation guidelines)
Precautions for use
All reagents should be considered as potentially hazardous. We
therefore recommend that this product is handled only by those
persons who have been trained in laboratory techniques and that it is
used in accordance with the principles of good laboratory practice.
Wear suitable protective clothing such as laboratory overalls, safety
glasses and gloves. Care should be taken to avoid contact with skin or
eyes. In the case of contact with skin or eyes wash immediately with
water. See material safety data sheet(s) for specic advice.
Preparation of buffers and solutions
Note: The quality of BSA is a critical parameter for the test
performance.
Phosphate buffered saline (PBS)
Reagents Quantity
NaCl 8.00 g
KCl 0.20 g
Na2HPO4 x 12 H2O 2.85 g
KH2PO40.20 g
H2O dest adjust to 1 liter
Wash buffer
Add 0.5 mL Tween 20 to 1 liter of PBS and mix well.
Assay buffer
Reagents Quantity
Bovine Serum Albumin (BSA) 5 g
Tween 20 0.5 mL
PBS adjust to 1 liter
Fixing buffer
Reagents Quantity
Sucrose 75 g
PBS adjust to 500 mL
Substrate solution
1:2 mixture of H2O2 and Tetramethylbenzidine
Stop solution
1M Phosporic Acid (H3PO4)
Preparation of the microwell plate
Coating
1. Coating antibody nal concentration is 1 µg/mL; 100 µL of the
coating solution is added to each well. Dilute the coating
antibody as following for one microtiter plate:
Reagents Volume
PBS 10.89 mL
Coating antibody (100 µg/mL) 0.11 mL
Coating solution (1 µg/mL) 11.00 mL
2. Immediately after coating, seal the plate with an adhesive lm
and store at 2°C to 8°C over night, allowing the binding process
to take place. Aspirate the contents of the wells and wash once
with 400 µL of Wash Buer according the washing procedure
described in the test protocol below (see “Test protocol“ on
page 2).
USER GUIDE
For Research Use Only. Not for use in diagnostic procedures.
Blocking and fixing
Blocking
Add 250 µL of Assay Buer to each well and incubate at room
temperature for 2 hours. Alternatively the plate may be blocked over
night at 2°C to 8°C. Blocked plates can be stored at 2°C to 8°C for up to
one week.
Fixing
To store the coated plates for longer than one week aspirate Assay
Buer and add 150 µL Fixing Buer to each well. Incubate for 1 hour
at room temperature. Aspirate Fixing Buer and dry plates for 2 hours
at 37°C. When sealed with desiccant, the plates can be stored at 2°C to
8°C for 2 months.
Preparation of immunological reagents
Note: Centrifuge vials before opening to collect contents.
Preparation of standard
1. Reconstitute rat IL-4 standard protein with deionized or distilled
water. Reconstitution volume is indicated on the vial label (nal
concentration of reconstituted standard = 2 ng/mL). Allow the
standard to reconstitute for 10-30 minutes. Mix well prior to
making dilutions.
2. The concentrated rat IL-4 standard must be diluted 1:10 with
Assay Buer just prior to use in a clean plastic test tube according
to the following dilution scheme:
Reagents Volume
conc. Standard Protein (2 ng/mL) 23 µL
Assay Buffer 207 µL
Standard Protein (200 pg/mL) 230 µL
3. Shake gently to mix. After usage remaining diluted standard
cannot be stored and has to be discarded.
4. Aliquot the reconstituted concentrated standard and store at
-20°C.
Preparation of Biotin-Conjugate
Dilute concentrated Biotin-Conjugate 1:1000 with Assay Buer before
use. Use within 30 minutes after preparation. For one microwell plate
dilute the stock reagents as follows:
Reagents Volume
conc. Biotin-Conjugate 5.5 µL
Assay Buffer 5494.5 µL
Biotin-Conjugate 5.5 mL
Preparation of Streptavidin-HRP
Dilute concentrated Streptavidin-HRP 1:10,000 with Assay Buer
before use. Use within 30 minutes after preparation. For one microwell
plate dilute the stock reagents as follows:
Reagents Volume
conc. Streptavidin-HRP 1.1 µL
Assay Buffer 10,998.9 µL
Streptavidin-HRP 11.0 mL
Test protocol
1. Wash blocked or blocked and xed plates twice with
approximately 400 µL Wash Buer per well, with thorough
aspiration of microwell contents between washes. Allow the Wash
Buer to sit in the wells for about 10 – 15 seconds before
aspiration. Take care not to scratch the surface of the microwells.
After the last wash step, empty wells and tap microwell strips on
absorbent pad or paper towel to remove excess Wash Buer. Use
the microwell plate immediately after washing. Alternatively
microwell plate can be placed upside down on a wet absorbent
paper for no longer than 15 minutes. Do not allow wells to dry.
2. Add 100 µL Sample Diluent in duplicate all standard wells.
Pipee 100 µL of diluted standard (see “Preparation of
standard“ on page 2), (concentration = 200 pg/mL) in duplicate
into well A1 and A2. Mix the contents of wells A1 and A2 by
repeated aspiration and ejection (concentration of standard 1, S1 =
100 pg/mL), and transfer 100 µL to wells B1 and B2, respectively
(see Figure 1). Take care not to scratch the surface of the
microwells. Continue this procedure 5 times, creating two serially
diluted columns of rat IL-4 standard dilutions ranging from 100.0
to 1.60 pg/mL. Discard 100 µL from the last microwells (G1, G2).
Final volume in all wells is 100 µL.
Transfer
100
µL
Reconstituted, diluted
Rat IL-4 Standard
S1
S2
S3
S4
-
S7
100 µL
Discard
100 µL
Fig. 1 Dilute standards - microwell plate
3. Add 100 µL of Sample Diluent in duplicate to blank wells.
4. Add 50 µL of Sample Diluent to the sample wells.
5. Add 50 µL of each sample in duplicate to the sample wells.
6. Prepare Biotin-Conjugate (see “Preparation of Biotin-
Conjugate“ on page 2)
7. Add 50 µL of prepared Biotin-Conjugate to all wells.
8. Cover with an adhesive lm and incubate at room temperature
(18°C to 25°C) for 2 hours, on a microplate shaker if available.
9. Prepare Streptavidin-HRP (see “Preparation of Streptavidin-
HRP“ on page 2).
10. Remove adhesive lm and empty wells. Wash microwells 3 times
according to point a. of the test protocol. Proceed immediately to
the next step.
11. Add 100 µL of diluted Streptavidin-HRP to all wells, including
the blank wells.
12. Cover with an adhesive lm and incubate at room temperature
(18°C to 25°C) for 1 hour, on a microplate shaker if available.
13. Remove adhesive lm and empty wells. Wash microwells 3 times
according to point a. of the test protocol. Proceed immediately to
the next step.
14. Pipee 100 µL of Substrate Solution to all wells.
15. Incubate the microwell strips at room temperature (18°C to 25°C)
for about 10 minutes. Avoid direct exposure to intense light.
Monitor the color development on the plate. The substrate
reaction should be stopped (see next point of this protocol) before
positive wells are no longer properly recordable.
Determination of the ideal time period for color development has
to be done individually for each assay.
Add the stop solution when the highest standard has developed a
dark blue color. Alternatively the color development can be
monitored on a plate reader at 620 nm. The substrate reaction
should be stopped as soon as Standard 1 has reached an OD of 0.9
– 0.95.
2
Rat IL-4 Matched Pair User Guide
16. Stop the enzyme reaction by quickly pipeing 100 µL of Stop
Solution into each well. It is important that the Stop Solution is
spread quickly and uniformly throughout the microwells to
completely inactivate the enzyme. Results must be read
immediately after the Stop Solution is added, or within one hour
if the microwell strips are stored at 2°C to 8°C in the dark.
17. Read absorbance of each microwell on a spectro-photometer
using 450 nm as primary wave length (you can use 620 nm as
reference wave length; 610 nm to 650 nm is acceptable). Blank the
plate reader according to the manufacturer's instructions by using
the blank wells. Determine the absorbance of both the samples
and the rat IL-4 standards.
Calculation of results
Calculate the average absorbance values for each set of duplicate
standards and samples. Duplicates should be within 20% of the
mean value.
Create a standard curve by ploing the mean absorbance for each
standard concentration on the y-axis, against the rat IL-4
concentration on the x-axis. Draw a best t curve through the
points of the graph (a 5-parameter curve t is recommended).
To determine the concentration of soluble rat IL-4 for each
sample, rst calculate the mean absorbance value for the
duplicate wells of the sample, then extend a horizontal line from
this point on the y-axis to the standard curve. At the point of
intersection, extend a vertical line to the x-axis and read the
corresponding rat IL-4 concentration.
If instructions in this protocol have been followed samples have
been diluted 1:2 (50 µL sample + 50 µL Sample Diluent), the
concentration read from the standard curve must be multiplied
by the dilution factor (x2).
Calculation of samples with a concentration exceeding that of
standard 1 may result in inaccurate, low rat IL-4 levels. Such
samples require further external predilution according to
expected rat IL-4 values with Sample Diluent in order to precisely
quantitate the actual rat IL-4 level.
Each testing facility should establish a control sample of known
rat IL-4 concentration and run this additional control with each
assay. If the values obtained are not within the expected range of
this control, the assay results may be invalid.
A basic understanding of immunoassay development and technical
experience in ELISA performance are conditional for the successful
use of this Module Set.
The protocol provided is just a guideline. The type of substrate as well
as all other reagents not included in the Module Set may inuence the
test characteristics.
Rat IL-4 module set characteristics
Specificity
The interference of circulating factors of the immune system was
evaluated by spiking these proteins at physiologically relevant
concentrations into a rat IL-4 positive serum. There was no cross
reactivity detected.
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products as set forth in the Life Technologies' General Terms and
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Bender MedSystems GmbH | Campus Vienna Biocenter 2 | 1030 Vienna, Austria
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The information in this guide is subject to change without notice.
DISCLAIMER: TO THE EXTENT ALLOWED BY LAW, THERMO FISHER SCIENTIFIC INC. AND/OR ITS AFFILIATE(S) WILL NOT BE LIABLE FOR SPECIAL, INCIDENTAL, INDIRECT,
PUNITIVE, MULTIPLE, OR CONSEQUENTIAL DAMAGES IN CONNECTION WITH OR ARISING FROM THIS DOCUMENT, INCLUDING YOUR USE OF IT.
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23 August 2019
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Thermo Fisher Scientific Rat IL-4 Matched Pair User guide

Type
User guide

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